Support continuation is being requested for a highly productive research in analytical glycobiology at Indiana University (IU). High-sensitivity quantitative measurements of glycoproteins and positive identification of their constituent glycan components are very important to many areas of biomedical research, particularly in a search for disease biomarkers. While our research during the last several years has made it possible to profile quantitatively N-glycans at considerably greater sensitivities than ever before, the proposed methodological improvements will focus increasingly on covering O-glycans using (a) greatly improved techniques of glycan release/MALDI mass spectrometry (MS); (b) derivatization and mass-shift strategies; and (c) profiling smaller O-glycans through GC/MS of permethylated derivatives. Additionally, significant improvements in preconcentration of trace glycoproteins from physiological fluids and tissues will be sought through the use of (a) surface-immobilized lectins; (b) surface-immobilized Protein L and selected antibodies; and (c) molecular size-based fractionation through novel capillary technologies. The surface- immobilized glycoprotein preconcentrators will utilize new types of highly porous silica materials prepared recently in a collaborating IU laboratory. New lectin types will augment this approach, focusing primarily on highly sialylated and hyperfucosylated glycans (both N- and O-types) that our laboratory has targeted as parts of cancer biomarkers under a complementary NCI grant. Our preliminary results with surface- immobilized Protein L provide indications that such procedures could rapidly sort out different immunoglobulin glycosylation patterns in very small volumes (few microliters) of physiological fluids. Alternatively, selected surface-immobilized antibodies to acute phase glycoproteins can be used to preconcentrate the fractions of diagnostic/prognostic interest. Properly treated extracts of tissues and cancer cell lines will als be investigated through a set of optimized procedures that will augment the analytical microscale methodologies developed for blood serum and plasma. With our laboratory's mass-spectrometric instrumentation and expertise in place, we will continue our emphasis on optimal biological sample treatment, glycoprotein fractionation, and preconcentration at microscale. PUBLIC HEALTH RELEVANCE: This proposal is a significant extension of the analytical glycobiology program at Indiana University, capitalizing on the development of highly sensitive measurements in glycomics and glycoproteomics accomplished during the past grant period. The proposal has a strong synergy with the NCI-supported program on cancer biomarkers with diagnostic and prognostic potential by the same PI. Proposed further methodological advances aim to (a) delve deeper into the intricacies of human glycome and extend the range of profiling physiological fluids; (b) identify the cancer and other disease-related aberrant glycosylated structures; and (c) connect the observed glycosylation differences to the proteins of interest.